Chronic pain is a major health and economic problem worldwide. Because the major analgesics (e.g., opioids) bind to receptors that are widely expressed throughout the central nervous system (CNS), dose-limiting adverse effects and significant risk of addiction and abuse present substantial barriers to their clinical use. Pain sensing neurons (a.k.a nociceptors) in dorsal root ganglion (DRG) play essential role in pain transmission by detecting painful signals in the periphery such as skin and viscera. Therefore, targeting molecules specifically expressed in nociceptors may offer an opportunity for pain-selective pharmacologic interventions. Our previous data have shown that Mrgs including mouse MrgC11 and human MrgX1 are specifically expressed in nociceptors in DRG and constitute an endogenous anti-pain pathway. Funded R03 grant allowed us to identify selective and potent MrgX1 allosteric agonists. Preliminary data showed that two allosteric agonists inhibited persistent pain in humanized MrgX1 mice. MrgX1 allosteric agonists may potentially become novel anti-chronic pain drugs with limited CNS-related side effects. In this proposal, we will improve and optimize MrgX1 allosteric agonists and test their analgesic efficacy in mice. Highly selective agonists, and for the purpose of this proposal, allosteric agonists, are needed in order to better understand the respective role of MrgX1 in these studies of chronic pain. We have recently discovered a novel class of allosteric agonists with potency on MrgX1 and selectivity against MrgX2. These selective allosteric agonists offer a unique opportunity to test the hypothesis presented in this proposal. In order to develop first-in-class, potent, selective and CNS penetrant MrgX1 allosteric agonists, we will optimize our lead scaffold such that the tool compound will possess the appropriate properties, a balance of potency, selectivity and DMPK. We will utilize an iterative medicinal chemistry approach which will enable all of the attributes to be evaluated in parallel. Once the tool compound(s) have been determined, we will then test these compounds in the humanized MrgX1 mouse model.